流动注射-化学发光法测定头孢氨苄

头孢氨苄在H2SO4溶液中降解后,其产物可在酸性条件下与Ce（Ⅳ）产生化学发光反应,罗丹明6G对该反应有较强的增敏作用。据此建立了流动注射－化学发光法测定头孢氨苄的新方法。该方法的线性范围为0.10－10.0mg/L检出限为0.06mg/L,相对标准偏差(n=11,c=1.00mg/L）为0.85。方法用于药物中头孢氨苄含量的测定,其结果与标准方法一致,回收率为96％－106％。     

Kinetic determination of cephalexin in drug formulations

A kinetic method for the accurate determination of cephalexin has been described. A solution of cephalexin is reacted with 5 x 10(-3)M cobalt(II) nitrate in 1 x 10(-3)M sodium hydroxide at 60 degrees C for a fixed time of 6 min, after which the absorbance of the reaction product is measured at 310 nm. The concentration of cephalexin is calculated by using the corresponding calibration equation for the fixed-time method. The method has been applied to proprietary drugs and the results were compared statistically with those given by the BP method. The determination of cephalexin by the fixed-concentration and rate-constant methods is feasible with the calibration equations obtained but the fixed-time method has been found to be more applicable.     

基于铜离子促水解反应高灵敏电化学检测鸡肉中头孢氨苄残留

利用纳米金( AuNPs)和L-半胱氨酸( Cys)修饰金电极( AuE),得到对Cu2+具有灵敏响应的电化学修饰电极( Cys/AuNPs/AuE)。在Cu2+存在时对头孢氨苄进行水解,通过方波伏安法测定水解液中剩余Cu2+的浓度,从而间接测得头孢氨苄的含量。对金电极的修饰条件、头孢氨苄的水解条件等进行了优化。在pH 4.5的HAc-NaAc缓冲液中,头孢氨苄在Cu2+存在时于沸水浴中水解25 min后,溶液中剩余Cu2+在Cys/AuNPs/AuE上有良好的电化学响应,还原峰电流差与头孢氨苄的浓度分别在0.0058~0.12μmol/L和0.12~2.9μmol/L范围内呈良好的线性关系,检出限为1.9 nmol/L(S/N=3)。用本方法对鸡肉样品中的头孢氨苄残留进行测定,结果表明,本方法简便快速、灵敏度高,适用于鸡肉等食品样品中头孢氨苄残留的测定。     

Differential pulse polarographic study of the degradation of cephalexin: Determination of Hydrogen Sulphide and other Degradation Products

Differential pulse polarography (d.p.p.) is used to study the degradation of cephalexin. Hydrogen sulphide, evolved during the degradation of cephalexin solutions, was removed continuously in a stream of nitrogen and determined periodically. Other electroactive degradation products were observed by d.p.p. of the degraded sample solutions. The degradation mechanism is highly dependent on pH, the initial concentration of cephalexin, temperature, the particular buffer used, and the presence of dissolved oxygen. The formation and degradation of the diketopiperazine derivative formed by intramolecular aminolysis, particularly at neutral pH, can be followed by means of its polarographic peak at -0.9 V (pH 7.4). Approximately half the total sulphur originally present in cephalexin is liberated as hydrogen sulphide at pH 7.4 at 37°C. Increasing the degradation temperature to 80°C and sweeping out the hydrogen sulphide with nitrogen increases the yield of a major product which gives a peak at -1.26 V. At pH 8.5 (80°C. 100 μg cephalexin ml^-1) the percentage of the sulphur evolved as hydrogen sulphide increases with time, and a peak appears at -0.96 V (probably 2-hydroxy-3-phenyl-6-methylpyrazine) which increases as the peak at -1.26 V becomes smaller. Other products formed under different conditions (concentration, pH, temperature) are reported. At pH 3 (80°C) only 8% conversion via intramolecular aminolysis and 5% evolution of total sulphur is indicated after four hours.     

Comparative study on the determination of cephalexin in its dosage forms by spectrophotometry and HPLC with UV-vis detection

This paper discusses the spectrophotometric determination of cephalexin as the intact cephalexin or as its acid-induced degradation product. Cephalexin can be determined in the range 1 × 10^–5–18 × 10^–5 M with relative standard deviations of 5-1%. The limits of quantitation and detection were 10^–5 and 0.3 × 10^–5 M, respectively. These procedures were compared with reversed-phase HPLC determination. No interference was observed in the presence of common pharmaceutical adjuvants. The H-point standard additions method was applied in order to correct for the possible presence of the cephalexin precursor, 7-aminocephalosporanic acid; this improves the selectivity of the UV-vis spectrophotometric method.     

Photometric extraction method for determination of cephalexin

Summary A photometric extraction method is developed for determination of cephalexin. Cephalexin is hydrolysed for 5 min in sulphuric acid medium on a steam-bath, the hydrolytic product forming a coloured compound with ninhydrin in 25% H₂SO₄. This compound is extracted with CHCl₃ and its absorbance measured at 520 nm. The reaction is selective — it is negative for the initial reagents in the cephalexin synthesis and for most other antibiotics. The only two antibiotics giving a similar reaction — ampicillin and cephalotin — do not interfere in the determination of cephalexin. The method is rather simple and permits rapid determination of cephalexin with satisfactory precision and accuracy — coefficient of variation 0.91–1,36%, relative error of the determination 0.66–1,19% (95% confidence limits).

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Zusammenfassung Eine extraktions-photometrische Bestimmungsmethode für Cephalexin wurde ausgearbeitet. Die Substanz wird 5 min auf dem Wasserbad in schwefelsaurem Milieu hydrolysiert. Das hydrolysierte Produkt bildet mit Ninhydrin in 25%iger Schwefelsäure eine gefärbte Verbindung. Diese wird mit Chloroform extrahiert und bei 520 nm gemessen. Die Reaktion ist selektiv; sie verläuft negativ bei den Ausgangsprodukten der CephalexinSynthese und bei den meisten anderen Antibioticis. Die einzigen beiden Antibiotika, die eine ähnliche Reaktion geben — Ampicillin und Cephalotin — stören die Bestimmung von Cephalexin nicht. Die Methode ist ziemlich einfach und ermöglicht die rasche Bestimmung von Cephalexin mit hinreichender Genauigkeit: der Variationskoeffizient beträgt 0,91–1,36%, der relative Fehler 0,66—1,19% (bei einer Vertrauensgrenze von 95%).

     

Kinetics and mechanisms of hydrolysis and aminolysis of thioxocephalosporins

The effect of replacing the beta-lactam carbonyl oxygen in cephalosporins by sulfur on their reactivity has been investigated. The second-order rate constant for alkaline hydrolysis of the sulfur analogue is 2-fold less than that for the natural cephalosporin. The thioxo derivative of cephalexin, with an amino group in the C7 side chain, undergoes beta-lactam ring opening with intramolecular aminolysis by a reaction similar to that for cephalexin itself. However, the rate of intramolecular aminolysis for the S-analogue is 3 orders of magnitude greater than that for cephalexin. Furthermore, unlike cephalexin, intramolecular aminolysis in the S-analogue occurs up to pH 14 with no competitive hydrolysis. The rate of intermolecular aminolysis of natural cephalosporins is dominated by a second-order dependence on amine concentration, whereas that for thioxocephalosporins shows only a first-order term in amine. The Bronsted beta(nuc) for the aminolysis of thioxo-cephalosporin is +0.39, indicative of rate-limiting formation of the tetrahedral intermediate with an early transition state with relatively little C-N bond formation.     

Spectrophotometric determination of cephalexin in dosage forms with imidazole reagent

A simple spectrophotometric method is proposed for the determination of cephalexin. The method involves acetylation of cephalexin with acetic anhydride in aqueous solution at pH 11.5 to yield alpha-acetamidocephalexin and subsequent measurement at 335 nm of alpha-acetamidocephalexin mercuric mercaptide. The method characterizes a newly developed, sensitive procedure for the determination of cephalexin in different pharmaceutical preparations. The effect of several reaction conditions were investigated. Beer's law was obeyed over the concentration range 30-340 mug/ml. The results compare favourably with those obtained by the official B.P. 1980 method.     

Synthesis of Cephalexin in Aqueous Medium with Carrier-bound and Carrier-free Penicillin Acylase Biocatalysts

The use of very high substrate concentrations favors the kinetically controlled synthesis of cephalexin with penicillin acylase (PA) not only by Michaelian considerations, but also because water activity is depressed, so reducing the rates of the competing reactions of product and acyl donor hydrolysis. Commercial PGA-450, glyoxyl agarose immobilized (PAIGA) and carrier-free cross-linked enzyme aggregates of penicillin acylase (PACLEA) were tested in aqueous media at concentrations close to the solubility of nucleophile and at previously determined enzyme to nucleophile and acid donor to nucleophile ratios. The best temperature and pH were determined for each biocatalyst based on an objective function considering conversion yield, productivity, and enzyme stability as evaluation parameters. Stability was higher with PAIGA and specific productivity higher with PACLEA, but best results based on such objective function were obtained with PGA-450. Yields were stoichiometric and productivities higher than those previously reported in organic medium, which implies significant savings in terms of costs and environmental protection. At the optimum conditions for the selected biocatalyst, operational stability was determined in sequential batch reactor operation. The experimental information gathered is being used for a technical and economic evaluation of an industrial process for enzymatic production of cephalexin in aqueous medium.     

Effect of Structural and Electrolytic Properties of Carboxyl-Containing Cellulose Cationites on the Sorption of Cephalexin from Aqueous Solutions

Sorption of antibiotic cephalexin from aqueous solutions by monocarboxyl cellulose (MCC) samples with various content of carboxyl groups and structure (the degree of crystallinity) is studied. It is disclosed that the sorption occurs mainly due to electrostatic interactions via the proton transfer from carboxyl group of MCC to the carboxylate ion of cephalexin. The sorption is characterized by Langmuir isotherms and described by the equation of localized stoichiometric sorption. Sorption of cephalexin increases with the content of carboxyl groups in the sorbent phase, which is explained by the peculiarities of the dissociation of MCC carboxyl groups. Structural characteristics of MCC samples do not substantially influence the sorption of antibiotic. Dependence of the sorption value on the pH of equilibrium solution has a maximum at pH 3.5, which is explained by the specific features of the electrolytic dissociation of cephalexin and MCC.     

Sorption removal of cephalexin by HNO3 and H2O2 oxidized activated carbons

Cephalexin’s traces within pharmaceutical effluents have toxic impact toward ecological and human health.Low-cost activated carbon derived from Trapa natans husk was oxidized with hydrogen peroxide and nitric acid,and tested for their ability to remove cephalexin from aqueous solutions.Oxidization with H2O2 showed negative effect on the cephalexin sorption,whereas HNO3 oxidization improved the adsorption.The cephalexin adsorption isotherms on the native and HNO3 oxidized carbons correlated well with the Freundlich equation while their kinetics followed the pseudo-second order model.The removal of cephalexin by the native and HNO3 oxidized carbons was found to be most favored at low ionic strength and strong acidic conditions.Based on the thermal and FTIR analyses,the interaction mechanisms of the interaction between cephalexin and the carbons were proposed.Electrostatic attraction,hydrophobic interaction and chemical bonding with surface functional groups were demonstrated as primary mechanisms for cephalexin removal.The nitrogen functionalities on the carbon surface were considered to be an important factor affecting the adsorption process.     

Application of thin layer and high performance liquid chromatography to the separation and determination of cephalexin in cephradine,in bulk powder and in pharmaceuticals

Summary A thin-layer chromatographic procedure for the quantitative determination of cephalexin in cephradine formulations is described. The thin-layer chromatography is carried out on a silica gel layer and the quantitation is by UV scanning of the chromatogram. The procedure was transferred to high-performance liquid chromatography on a microparticulate silica gel column with a similar eluent system. The cephalexin content of a few commercial cephradine samples is determined by using these techniques. The results of these procedures are compared to those obtained with high-performance liquid chromatography on a C 18 reverse phase column.     

Simultaneous Determination of Cephradine,L‐Arginine,and Cephalexin in Cephradine for Injection by Capillary Zone Electrophoresis

Abstract

Applying capillary zone electrophoresis (CZE) to separate the components of Cephradine for Injection: cephradine, and L‐arginine, as well as cephalexin, which is the degradation product of cephradine was studied. The best results were achieved with background electrolyte consisting of 50 mM disodium hydrogen phosphate buffer at pH 6.5 and an applied voltage of 20 kV in a bare fused‐silica capillary. The samples were injected at 50 mbar for 4 s. The capillary temperature was 25°C and the UV detection was performed at a wavelength of 195 nm. Histidine was used as internal standard (IS) to ensure acceptable precision data. The linear ranges of cephradine, L‐arginine, and cephalexin were 93.8–6255.6 µg/mL, 47.9–3195.2 µg/mL, and 6.1–405.4 µg/mL, respectively. Quantitative parameters such as accuracy, precision, limit of detection (LOD), and limit of quantitation(LOQ) were all established in CZE mode.     

Spectrophotometric Determination of Cephalosporins in Pure Form and in Pharmaceutical Preparations

Abstract

A spectrophotometric determination is described for cephalosporins, offering adequate sensitivity and good precision. The procedure applies successfully to a wide variety of cephalosporins, also in pharmaceutical preparations: cephalothin, cefacetrile, cephapirin, cefotaxime, ceftizoxime, cephaloridine, cefazolin, cefamandole nafate, cephalexin, cefadroxil, cefoxitin and cefuroxime. The method employs a reaction with ammonium molybdate in sulphuric acid medium. The antibiotic is heated at 91.5°C for 15 min and the absorbance of the coloured product is measured at 670 nm against a reagent blank treated similarly. Beer's law is obeyed up to 125 to 150 μg of cephalosporin in the 5-ml final solution. The effect of reagent concentration and reaction conditions are discussed.     

A reversed phase high-performance liquid chromatography method for determination of cephalexin in human plasma

A reversed-phase HPLC method for the analysis of cephalexin (7-[(aminophenylacetyl)amino]-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid) by isocratic separation is described. A comparison is made between the ultrafiltration land the extraction procedure developed in our laboratory. The extraction procedure, based on the deproteinization of plasma with perchloric acid followed by the extraction with dichloroethane and separation of cephalexin on a reversed phase column, gave better recovery. Quantitative validation of our method has been performed by an external standard technique. The relative standard deviations were between 1.1 and 1.9% in the within-day assay and between 1.2 and 2.2% in the inter-day assay. The limit of detection was 0.14ug/ml and the limit of quantification was 0.28ug/ml. This method is rapid, sensitive, simple, and reproducible. The LC MS/MS technique was successfully used for identification and quantification of cephalexin in human plasma samples.     

Analysis of cephalexin from canine skin biopsy by liquid chromatography with ultraviolet-visible photodiode-array detection

A sensitive and selective ion-paired liquid chromatographic method with UV-VIS photodiode-array detection was developed to measure cephalexin in skin biopsy samples. The method involved a sonication of minced canine skin with ethanol-acetonitrile-water (30:20:50, v/v/v) and ultrafiltration of received extract through 10,000 daltons. Separation of cephalexin from other components was by liquid chromatography using a reversed-phase column which was eluted with an ion-paired acetonitrile-water solution. Detection was achieved with a UV-VIS photodiode-array detector scanning from 230 to 320 nm. Cephalexin in the eluate was quantitated at its wavelength maximum of 260 nm. The evaluation of chromatographic peak homogeneity was performed by absorbance ratios, contour maps, first-derivative spectra and a three-dimensional spectrochromatogram. Additionally, the cephalexin peak identity was confirmed by liquid chromatography-mass spectrometry.     

A new derivatization procedure for the determination of cephalexin with 1,2-naphthoquinone 4-sulphonate in pharmaceutical and urine samples using solid-phase extraction cartridges and UV–visible detection

The present report shows how to derivatize cephalexin with 1,2-naphthoquinone-4-sulphonate (NQS) into solid-phase extraction cartridges (C₁₈) using UV–visible detection. Optimum conditions for this new procedure are: hydrogen carbonate/carbonate buffer pH=10.5, 5 min reaction time at 25°C and NQS concentration of 7.1×10⁻³ mol l⁻¹. The accuracy and the precision of the method were tested. The procedure was used to measure cephalexin in pharmaceutical and urine samples. The results obtained were contrasted with those reported by UV-spectrophotometric and HPLC methods for pharmaceutical samples and with HPLC method for urine samples. The H-point standard additions method was used to measure cephalexin in pharmaceutical samples, and the generalized H-point standard additions method was used to measure cephalexin in urine samples.     

Differential pulse polarographic determination of cephalexin based on the catalytic pre-wave of nickel(II)

A nickel-catalytic pre-peak at ?0.72 V vs. Ag/AgCl, is caused by cephalexin in sodium acetate (pH 8.3) medium. It is used to determine 2.4 × 10^?7–6 × 10^?6 M cephalexin; the relative standard deviation is 4% for 0.88 μg ml^?1 cephalexin. Other cephalosporins behave similarity; some amino acids can be tolerated in moderate amounts.     

药物头孢Ⅳ号和氨苄青霉素及其生物合成前体的毛细管区带电泳分离

 研究了同分异构体药物头孢Ⅳ号和氨苄青霉素及其目标生物合成前体三肽Ｄ－苯丙氨酸－Ｌ－半胱氨酸－Ｄ－缬氨酸、Ｌ－苯丙氨酸－Ｌ－半胱氨酸－Ｄ－缬氨酸的毛细管区带电泳分离。结果表明，采用含１２ｍｍｏｌ／Ｌ二甲基β－环糊精（ＤＭ－β－ＣＤ）的５０ｍｍｏｌ／ＬＴｒｉｓ－Ｈ３ＰＯ４缓冲溶液（ｐＨ２．３）既可将头孢Ⅳ号和氨苄青霉素分离，又可将Ｄ－苯丙氨酸－Ｌ－半胱氨酸－Ｄ－缬氨酸、Ｌ－苯丙氨酸－Ｌ－半胱氨酸－Ｄ－缬氨酸分离。     

Recombinant alpha-amino ester acid hydrolase from Xanthomonas rubrilineans VKPM B-9915 is a highly efficient biocatalyst of cephalexin synthesis

Recombinant, as well as native alpha-amino acid ester hydrolase from Xanthomonas rubrilineans VKPM B-9915 (XrAEH, EC 3.1.1.43), was tested for synthesis of amino-beta-lactam antibiotic cephalexin. It was shown that the recombinant enzyme r-XrAEH produced by Escherichia coli VKPM B-11246 is more efficient in comparison with the native enzyme wt-XrAEH prepared from mutant strain Xanthomonas rubrilineans VKPM B-9915. When r-XrAEH was used as a biocatalyst, addition of ethylene glycol (33 vol %) to the reaction medium improved the yield from 70 to 95%. During synthesis of cephalexin under optimal conditions in the case of the native enzyme wt-XrAEH the cephalexin yield was 85%, in contrast to r-XrAEH where it was 95%. Furthermore, unlike native wt-XrAEH enzymes, preparations of recombinant r-XrAEH do not possess beta-lactamase side activity.